Evaluation of Inhibitory Effect and Mechanism of Euphorbia Factor L3 against Phytophthora capsici

Phytophthora capsici is a highly destructive phytopathogenic oomycete with a broad host range and is responsible for tremendous losses. Euphorbia factor L3 (EFL3) is a natural plant-derived compound that has been widely studied in medicine and cosmetic applications. In this study, the sensitivity of 105 P. capsici isolates to EFL3 was determined, and the biological activity and physiological effects of EFL3 against P. capsici were investigated. The median effective concentration (EC50) values for EFL3 inhibition mycelial growth and spore germination ranged from 0.66 to 8.94 μg/mL (mean, 2.96 ± 0.91 μg/mL) and 1.63 to 13.16 μg/mL (mean, 5.30 ± 1.64 μg/mL), respectively. EFL3 treatment resulted in cell wall and cell membrane damage of P. capsici, which was revealed by morphological and ultrastructural observations, propidium iodide (PI) and calcofluor white (CFW) staining, and measurements of relative conductivity as well as malondialdehyde (MDA) and glycerol contents. In addition, the contents of phospholipid and cellulose, which are the major components of cell membrane and cell wall, were significantly reduced following EFL3 treatment. Furthermore, EFL3 provided protective as well as curative efficacies against P. capsici on detached tomato leaves and pepper seedlings in vivo. These data show that EFL3 exhibits strong inhibitory activity against P. capsici, thereby suggesting that it could be an effective alternative for controlling P. capsici-induced diseases.


Introduction
Phytophthora capsici Leonian is a notoriously harmful pathogen that can infect more than 60 species of vegetable crops (e.g., pepper, tomato, eggplant, and some melon crops) in the Solanaceous, Cucurbitaceous, and Fabaceous families [1][2][3]. Typical symptoms caused by P. capsici on vegetables include stem and fruit rot, wilting, stunting, damping off, and plant death, along with stem and leaf blight [4,5]. It is estimated that P. capsici-induced diseases are responsible for at least $200 million in losses each year [6]. As a soilborne pathogenic oomycete, P. capsici can survive in the soil for a long time, which leads to difficulties in prevention and control [1,2].
At present, P. capsici control mainly depends on synthetic chemical fungicides, such as mefenoxam and metalaxyl. However, long-term use of the existing fungicides has led to the emergence of fungicide-resistant P. capsici, which has resulted in control failures [7,8]. In addition, there is considerable concern regarding the adverse effects that some chemical fungicides have on the environment and food safety [9]. Therefore, there is an urgent need to develop natural, safe, and effective antifungal substances for P. capsici control. In this sense, plant natural products with antifungal properties are an ideal choice because of their low toxicity and environmental sustainability. Many plant natural products have been reported to possess promising antifungal properties and are recommended for use in controlling plant pathogens. For example, poacic acid inhibits the growth of because of their low toxicity and environmental sustainability. Many plant natural products have been reported to possess promising antifungal properties and are recommended for use in controlling plant pathogens. For example, poacic acid inhibits the growth of the fungi Sclerotinia sclerotiorum and Alternaria solani as well as the oomycete Phytophthora sojae [10]. Further, hinokitiol efficiently suppresses the growth of Alternaria alternata and Botrytis cinerea in postharvest disease measures [11]. More recently, we reported that 10-deacetylbacatin III exhibits effective inhibition against various oomycetes [12].
Euphorbia factor L3 (EFL3, Figure 1A) is a lathyrane diterpenoid with a 5/11/3-membered ring system [13,14]. It is a natural product isolated mainly from numerous plants from the Euphorbiaceae family, genus Euphorbia, which are mostly distributed in Africa, Southeast Asia, and Central to South America [15]. EFL3 is one of the main diterpenoids in the seeds of E. lathyris, which accounts for 40% of the total diterpenoids in the seeds [16]. The high content of EFL3 facilitates its large-scale production and application. Recent studies have revealed that EFL3 has a variety of biological activities, such as anti-inflammatory, antitumor, reversing multidrug resistance of tumor cells, and skin whitening [14,[17][18][19]. However, there have been no studies into the potential of EFL3 as a control agent against phytopathogens and its potential mechanism(s) of action. Therefore, the aims of this study were to (i) investigate the baseline sensitivity of EFL3 against P. capsici collected from Jiangsu, Shandong, and Anhui Provinces in China; (ii) explore the mechanism(s) of action of EFL3 by biochemical and physiological analyses; and (iii) determine the control effect of EFL3 against P. capsici on detached tomato leaves and by pot experiments. The results of this study may provide an efficient alternative approach for controlling P. capsici-induced diseases. Therefore, the aims of this study were to (i) investigate the baseline sensitivity of EFL 3 against P. capsici collected from Jiangsu, Shandong, and Anhui Provinces in China; (ii) explore the mechanism(s) of action of EFL 3 by biochemical and physiological analyses; and (iii) determine the control effect of EFL 3 against P. capsici on detached tomato leaves and by pot experiments. The results of this study may provide an efficient alternative approach for controlling P. capsici-induced diseases.

Baseline Sensitivity of P. capsici to EFL 3 by Mycelial Growth and Spore Germination
The sensitivity of P. capsici to EFL 3 was determined using 105 isolates collected from different areas of Jiangsu, Anhui, and Shandong Provinces in 2020 and 2021 (Table S1). The individual EC 50 values of EFL 3 for inhibiting mycelial growth ranged from 0.66 to 8.94 µg/mL, with an average value of 2.96 ± 0.91 µg/mL. EFL 3 -resistant populations to EFL 3 were not identified among these strains, and the frequency distribution of these EC 50 values was unimodal over a sensitive range ( Figure 1B). The range-of-variation factor (maximum/minimum EC 50 value) was 13.55.
To further determine the effect of EFL 3 on the secondary infection of P. capsici, the sensitivity of EFL 3 against spore germination of P. capsici isolates was tested. The results showed that the individual EC 50 values for EFL 3 -mediated inhibition of spore germination ranged from 1.63 to 13.16 µg/mL, with an average value of 5.30 ± 1.64 µg/mL, which was approximately 1.8 times the corresponding value for mycelial growth. The frequency distribution of EC 50 values also formed a unimodal curve, and the range-of-variation factor was 8.07 ( Figure 1C). These results indicate that EFL 3 has a strong inhibitory effect on mycelial growth and spore germination of P. capsici. In addition, among the 105 isolates of P. capsici, a metalaxyl-resistant strain (TA54, isolated from a host pepper plant in Tai'an, Shandong Province, China) was selected for studies on inhibitory activity and EFL 3 mechanism.

EFL 3 Altered P. capsici Morphology and Ultrastructure
The changes in mycelial morphology of P. capsici were clearly shown by optical microscopy (OM) observations. The mycelia of LT263 (the standard strain) and TA54 (a metalaxyl-resistant strain) without exposure to EFL 3 were uniform, regular, and robust, with a smooth surface and constant diameter. However, after treatment with 5 µg/mL of EFL 3 , the mycelia became swollen and contorted and produced more abnormal branches. Moreover, after treatment with high concentration (10 µg/mL) of EFL 3 , the mycelia became more obviously shrunken and distorted, and the offshoots at the top became shorter ( Figure 2).
The individual EC50 values of EFL3 for inhibiting mycelial growth ranged from 0.66 to 8.9 µg/mL, with an average value of 2.96 ± 0.91 µg/mL. EFL3-resistant populations to EFL were not identified among these strains, and the frequency distribution of these EC50 val ues was unimodal over a sensitive range ( Figure 1B). The range-of-variation factor (maxi mum/minimum EC50 value) was 13.55.
To further determine the effect of EFL3 on the secondary infection of P. capsici, th sensitivity of EFL3 against spore germination of P. capsici isolates was tested. The result showed that the individual EC50 values for EFL3-mediated inhibition of spore germinatio ranged from 1.63 to 13.16 µg/mL, with an average value of 5.30 ± 1.64 µg/mL, which wa approximately 1.8 times the corresponding value for mycelial growth. The frequency dis tribution of EC50 values also formed a unimodal curve, and the range-of-variation facto was 8.07 ( Figure 1C). These results indicate that EFL3 has a strong inhibitory effect on my celial growth and spore germination of P. capsici. In addition, among the 105 isolates of P capsici, a metalaxyl-resistant strain (TA54, isolated from a host pepper plant in Tai'an Shandong Province, China) was selected for studies on inhibitory activity and EFL3 mech anism.

EFL3 Altered P. capsici Morphology and Ultrastructure
The changes in mycelial morphology of P. capsici were clearly shown by optical mi croscopy (OM) observations. The mycelia of LT263 (the standard strain) and TA54 (a met alaxyl-resistant strain) without exposure to EFL3 were uniform, regular, and robust, wit a smooth surface and constant diameter. However, after treatment with 5 µg/mL of EFL the mycelia became swollen and contorted and produced more abnormal branches. More over, after treatment with high concentration (10 µg/mL) of EFL3, the mycelia becam more obviously shrunken and distorted, and the offshoots at the top became shorter (Fig  ure 2). Transmission electron microscopy (TEM) observations were further performed t evaluate the effect of EFL3 on mycelial ultrastructure of P. capsici. The mycelia of LT26 and TA54, grown in the absence of EFL3, contained a uniform cytoplasmic matrix an intact cellular organelles. The plasma membrane and cell wall were smooth and intact. B Transmission electron microscopy (TEM) observations were further performed to evaluate the effect of EFL 3 on mycelial ultrastructure of P. capsici. The mycelia of LT263 and TA54, grown in the absence of EFL 3 , contained a uniform cytoplasmic matrix and intact cellular organelles. The plasma membrane and cell wall were smooth and intact. By contrast, the normal ultrastructure of P. capsici was destroyed in the EFL 3 treatment group. After exposure to 5 µg/mL of EFL 3 , the thickness of the plasma membrane and cell wall decreased, the vasculature increased, and the cellular organelles began to degrade. High concentrations of EFL 3 (10 µg/mL) caused greater damage to the mycelial ultrastructure. The plasma membrane and cell wall ruptured; cell vacuolation was greater; and cellular organelles, such as nucleus and mitochondria, completely disappeared ( Figure 3). These data suggest that EFL 3 causes severe disruption of morphology and ultrastructure of P. capsici, leading to cell death. decreased, the vasculature increased, and the cellular organelles began to degrade. High concentrations of EFL3 (10 µg/mL) caused greater damage to the mycelial ultrastructure. The plasma membrane and cell wall ruptured; cell vacuolation was greater; and cellular organelles, such as nucleus and mitochondria, completely disappeared ( Figure 3). These data suggest that EFL3 causes severe disruption of morphology and ultrastructure of P. capsici, leading to cell death.

EFL3 Affected the Cell Membrane Permeability of P. capsici
The effect of EFL3 on the cell membrane permeability of P. capsici was investigated by measuring the extracellular conductivity as well as malondialdehyde (MDA) and glycerol contents of P. capsici. As shown in Figure 4A,D, after treatment with EFL3, the relative conductivities of LT263 and TA54 were always higher than the untreated control. The relative conductivity increased with increasing EFL3 concentrations and times. After incubation for 120 min, the relative conductivities of LT263 and TA54 in 10 µg/mL of EFL3 treatment group were 4.18 and 3.81 times higher, respectively, than that of the untreated control ( Figure 4A,D).

EFL 3 Affected the Cell Membrane Permeability of P. capsici
The effect of EFL 3 on the cell membrane permeability of P. capsici was investigated by measuring the extracellular conductivity as well as malondialdehyde (MDA) and glycerol contents of P. capsici. As shown in Figure 4A,D, after treatment with EFL 3 , the relative conductivities of LT263 and TA54 were always higher than the untreated control. The relative conductivity increased with increasing EFL 3 concentrations and times. After incubation for 120 min, the relative conductivities of LT263 and TA54 in 10 µg/mL of EFL 3 treatment group were 4.18 and 3.81 times higher, respectively, than that of the untreated control ( Figure 4A,D).  As illustrated in Figure 4B,E, treatment with EFL3 resulted in a remarkable increase in the MDA content of both LT263 and TA54 compared to the control. This response was also dose dependent. Similar results were observed for glycerol content. The addition of EFL3 induced a significant increase in the glycerol content of LT263 and TA54, while the As illustrated in Figure 4B,E, treatment with EFL 3 resulted in a remarkable increase in the MDA content of both LT263 and TA54 compared to the control. This response was also dose dependent. Similar results were observed for glycerol content. The addition of EFL 3 induced a significant increase in the glycerol content of LT263 and TA54, while the levels in the controls remained stable during the whole period. In addition, the glycerol content also increased as the EFL 3 concentration increased ( Figure 4C,F). These results suggest that EFL 3 may target cell membrane permeability of P. capsici.

EFL 3 Destroyed the Cell Membrane and Cell Wall Integrity of P. capsici
To further explore the potential mechanisms by which EFL 3 exhibits antioomycete activity, propidium iodide (PI) and calcofluor white (CFW) straining were used to determine cell membrane and cell wall integrity. PI is a fluorescent dye that is often used to indicate cell membrane damage [20]. As shown in Figure 5, the mycelia of LT263 and TA54 in the untreated control showed no detectable fluorescence, presumably because PI was unable to penetrate mycelia with an intact membrane. However, the mycelia showed a bright red fluorescence after exposure to EFL 3 .
cules 2023, 27, x FOR PEER REVIEW Figure 5. Effect of EFL3 on the cell membrane integrity of P. capsici. The mycelia LT263 and TA54 were stained with propidium iodide (PI) and observed unde croscope. The scale bar represents 50 µm.
CFW is a fluorescent brightener that has strong affinity to cellulose various plants and oomycetes [21]. Here, the effect of EFL3 on cell wall lyzed by CFW staining. As shown in Figure 6, EFL3 treatment signific cellulose distribution of both LT263 and TA54. The blue fluorescence EFL3 treatment group was weaker than the untreated control. All the that EFL3 impairs cell membrane and cell wall integrity of P. capsici. CFW is a fluorescent brightener that has strong affinity to cellulose in the cell wall of various plants and oomycetes [21]. Here, the effect of EFL 3 on cell wall integrity was analyzed by CFW staining. As shown in Figure 6, EFL 3 treatment significantly affected the cellulose distribution of both LT263 and TA54. The blue fluorescence of mycelia in the EFL 3 treatment group was weaker than the untreated control. All these results indicate that EFL 3 impairs cell membrane and cell wall integrity of P. capsici.
CFW is a fluorescent brightener that has strong affinity to cellulose in the cell wall of various plants and oomycetes [21]. Here, the effect of EFL3 on cell wall integrity was analyzed by CFW staining. As shown in Figure 6, EFL3 treatment significantly affected the cellulose distribution of both LT263 and TA54. The blue fluorescence of mycelia in the EFL3 treatment group was weaker than the untreated control. All these results indicate that EFL3 impairs cell membrane and cell wall integrity of P. capsici.

EFL 3 Affected the Phospholipid and Cellulose Contents of P. capsici
Phospholipids are the main components of the oomycete cell membrane, which is very important for maintaining cell membrane integrity and function [22,23]. In this study, the phospholipid content in the mycelia of LT263 and TA54 were detected. As shown in Figure 7A,C, the phospholipid content of P. capsici was significantly lower than that of the control group after exposure to different concentrations of EFL 3 . Compared to the untreated control, treatment with EFL 3 at 1.25, 2.5, 5, and 10 µg/mL resulted in a decrease in the phospholipid content of LT263 and TA54 by 16

EFL3 Affected the Phospholipid and Cellulose Contents of P. capsici
Phospholipids are the main components of the oomycete cell membrane, which is very important for maintaining cell membrane integrity and function [22,23]. In this study, the phospholipid content in the mycelia of LT263 and TA54 were detected. As shown in Figure 7A,C, the phospholipid content of P. capsici was significantly lower than that of the control group after exposure to different concentrations of EFL3. Compared to the untreated control, treatment with EFL3 at 1.25, 2.5, 5, and 10 µg/mL resulted in a decrease in the phospholipid content of LT263 and TA54 by 16  Cellulose acts as the structural backbone of the oomycete cell wall [24]. To further establish the effect of EFL3 on the cell wall, the cellulose content of P. capsici was also determined. The cellulose content of LT263 and TA54 incubated with 1.25, 2.5, 5, and 10 µg/mL of EFL3 were 14.21, 10.28, 8.67, and 5.23 mg/g and 15.28, 10.90, 7.31, and 5.04 mg/g, Cellulose acts as the structural backbone of the oomycete cell wall [24]. To further establish the effect of EFL 3 on the cell wall, the cellulose content of P. capsici was also determined. The cellulose content of LT263 and TA54 incubated with 1.25, 2.5, 5, and 10 µg/mL of EFL 3 were 14.21, 10.28, 8.67, and 5.23 mg/g and 15.28, 10.90, 7.31, and 5.04 mg/g, respectively, which were significantly lower than that of the untreated control (23.54 and 24.42 mg/g) ( Figure 7B,D). Hence, EFL 3 may reduce the phospholipid and cellulose contents of P. capsici to further affect the integrity of the cell membrane and cell wall.

Curative and Protective Activities of EFL 3 against P. capsici
To understand the control efficacy of EFL 3 against P. capsici, the protective and curative activities of EFL 3 on detached leaves of tomato were determined. Tomato leaf inoculation assays showed that EFL 3 exhibited strong control efficacy on the diseases caused by P. capsici. As shown in Figure 8A, EFL 3 reduced the symptoms caused by LT263 and TA54 in a dose-dependent manner. The curative and protective efficacies of EFL 3 were 50.64 and 45.76% for LT263 and 19.54 and 21.65% for TA54, respectively, after treatment with 50 µg/mL of EFL 3 . When the concentration of EFL 3 reached 200 µg/mL, its curative and protective efficacies were 92.47 and 100% for LT263 and 82.68 and 95.49% for TA54, respectively. Moreover, the curative and protective efficacies of 200 µg/mL metalaxyl against LT263 were 98.86 and 96.52%. In contrast, the curative and protective efficacies of 200 µg/mL metalaxyl against TA54 was much lower (only 14.70 and 23.51%, respectively; Figure 8B,C). Given that P. capsici is a soilborne pathogen, to further clarify the control efficacy of EFL3 against P. capsici, additional pot experiments were conducted on pepper seedlings. Similarly, as shown in Figure 9A, the disease symptoms on pepper seedlings caused by P. capsici were significantly inhibited by EFL3 treatment. After exposure to 100 µg/mL of EFL3, the curative and protective efficacies were 51.36 and 58.52% for LT263 and 48.52 and 57.36% for TA54, respectively. When the concentration of EFL3 reached 200 µg/mL, the curative and protective efficacies of EFL3 were 76.73 and 87.64% for LT263 and 83.54 and 92.42% for TA54, which was consistent with the results of the detached leaf assay. Notably, the protective activity of EFL3 against P. capsici was always superior than the curative activity within the same treatment concentration (Figure 9B,C). Overall, these results suggest that EFL3 may be a promising fungicide against diseases caused by P. capsici. Given that P. capsici is a soilborne pathogen, to further clarify the control efficacy of EFL 3 against P. capsici, additional pot experiments were conducted on pepper seedlings. Similarly, as shown in Figure 9A, the disease symptoms on pepper seedlings caused by P. capsici were significantly inhibited by EFL 3 treatment. After exposure to 100 µg/mL of EFL 3 , the curative and protective efficacies were 51.36 and 58.52% for LT263 and 48.52 and 57.36% for TA54, respectively. When the concentration of EFL 3 reached 200 µg/mL, the curative and protective efficacies of EFL 3 were 76.73 and 87.64% for LT263 and 83.54 and 92.42% for TA54, which was consistent with the results of the detached leaf assay. Notably, the protective activity of EFL 3 against P. capsici was always superior than the curative activity within the same treatment concentration ( Figure 9B,C). Overall, these results suggest that EFL 3 may be a promising fungicide against diseases caused by P. capsici. EFL3 against P. capsici, additional pot experiments were conducted on pepper seedlings. Similarly, as shown in Figure 9A, the disease symptoms on pepper seedlings caused by P. capsici were significantly inhibited by EFL3 treatment. After exposure to 100 µg/mL of EFL3, the curative and protective efficacies were 51.36 and 58.52% for LT263 and 48.52 and 57.36% for TA54, respectively. When the concentration of EFL3 reached 200 µg/mL, the curative and protective efficacies of EFL3 were 76.73 and 87.64% for LT263 and 83.54 and 92.42% for TA54, which was consistent with the results of the detached leaf assay. Notably, the protective activity of EFL3 against P. capsici was always superior than the curative activity within the same treatment concentration ( Figure 9B,C). Overall, these results suggest that EFL3 may be a promising fungicide against diseases caused by P. capsici.

Discussion
P. capsici is a highly destructive phytopathogenic oomycete with a broad host range [1][2][3]. At present, fungicide application is still the main method for controlling diseases caused by P. capsici. The long-term use of conventional fungicides, however, has increased the development of fungicide-resistant pathogens [7,8]. For these reasons, chemical substitutes with different modes of action and high inhibitory activity are urgently needed. EFL 3 is a lathyrane diterpenoid, which is a natural product mainly isolated from Euphorbiaceae plants [13,14]. In recent years, the medical and cosmetic values of EFL 3 have been extensively reported, but its antifungal activities against phytopathogens have not been reported yet [14,[17][18][19]. In this study, we evaluated EFL 3 -mediated control of diseases caused by P. capsici.
Baseline sensitivity is very important for monitoring the occurrence of fungicide resistance. Hence, it is necessary to determine the baseline sensitivity of the pathogens to fungicide before its legal use [25]. In this study, baseline sensitivity to EFL 3 was first determined by measuring the sensitivity of 105 isolates of P. capsici collected from Jiangsu, Anhui, and Shandong Provinces in China. The baseline sensitivity curves of EFL 3 were unimodal with average EC 50 values of 2.96 ± 0.91 and 5.30 ± 1.64 µg/mL for inhibition of mycelial growth and spore germination, respectively, while the range-of-variation factors were 13.55 and 8.07, respectively. The low EC 50 values and narrow range-of-variation factors suggest that no EFL 3 -resistant isolates were found in our study. Furthermore, the average EC 50 values for EFL 3 were lower than the previously published EC 50 values of other plant-derived natural products, such as cinnamaldehyde and esculetin, against P. capsici [26,27]. These data show that EFL 3 has a strong inhibitory effect on mycelial growth and spore germination of P. capsici.
Given the excellent inhibitory effect of EFL 3 against P. capsici, we evaluated the physiological and biochemical effects of EFL 3 against P. capsici to explore antifungal mechanisms. The antifungal activity of plant-derived terpenoids has been partly attributed to their destructive effects on pathogen cell wall and cell membrane. For example, thymol was reported to lead to the loss of cell morphology, irregular shrinkage, and even serious fracture of hyphae of Botrytis cinerea [28]. In this study, we found that EFL 3 damaged the morphological and ultrastructural characteristics of P. capsici. In addition, EFL 3 treatment caused increased electrical conductivity and upregulated MDA and glycerol contents. Furthermore, using PI and CFW straining, we found that EFL 3 severely disrupted the cell membrane and cell wall integrity of P. capsici, which eventually led to cell death. Taken together, these results suggest that the antifungal mechanism of EFL 3 may be attributed to its destructive potential in the cell membrane and cell wall.
Phospholipids are major components of the oomycete cell membrane, which is very important for maintaining cell membrane integrity and function [22,23]. Cellulose acts as the structural backbone of the oomycete cell wall [24]. Many fungicides have been reported to destroy the cell membrane or cell wall by inhibiting their main structural components. For example, clotrimazole and itraconazole achieved their fungicidal effects against citrus green mold by causing a decrease in ergosterol content, which subsequently affected cell membrane integrity and function [29,30].  showed that carvacrol significantly decreased the total lipid and ergosterol contents to disrupt the cell membrane in the hyphae of Botryosphaeria dothidea [31]. In our study, phospholipid and cellulose contents decreased significantly following EFL 3 treatment, which would explain the observed destruction of the cell membrane and cell well. However, the mechanism by which EFL 3 causes the decrease in phospholipid and cellulose contents in P. capsici remains unclear. Thus, further investigations are required to elucidate this phenomenon.
Fungicides with both protective and curative effects can effectively control disease occurrence and prevalence [32]. In this study, we investigated the protective and curative activities of EFL 3 against P. capsici on detached tomato leaves and by pot experiments. The results indicated that EFL 3 displayed both protective and curative activities against P. capsici on detached tomato leaves and pepper seedlings. Moreover, the protective activity of EFL 3 was better than the curative activity at the same concentration on pepper seedlings. This suggests that EFL 3 should be used prophylactically or in the early stage of disease for better control. In addition, the control effect of EFL 3 against P. capsici seemed to be more effective than that of other plant-derived natural products, such as liquiritin and cinnamaldehyde [27,32]. Our data suggest that EFL 3 has the potential to be an excellent alternative fungicide to control diseases caused by P. capsici. To further confirm the control efficacy of EFL 3 against P. capsici, field experiments will be carried out in the near future. The antifungal mechanism of EFL 3 may be attributed to its destructive potential in the cell membrane and cell wall. Because the composition of the cell membrane and cell wall is similar in plants and oomycetes [33,34], EFL 3 may have some effects on nontarget organisms (plants). In this study, we use the inoculation assay on both tomato leaves and pepper seedlings by treating them with different concentrations of EFL 3 . However, as shown in Figures 8 and 9, there was no visible effect of EFL 3 on tomato leaves and pepper seedlings. The effect of EFL 3 on the fruit quality of tomato and pepper will be determined in further studies.

Chemicals
EFL 3 was isolated from Euphorbia lathyris as previously reported [35]. Briefly, seeds of E. lathyris (2 kg) were refluxed with 95% (v/v) aqueous ethanol. After filtration, the solution was concentrated under reduced pressure to make a residue (230.4 g), which was suspended in distilled water and successively partitioned with petroleum ether, dichloromethane (DCM), and ethyl acetate. The DCM extract was separated and repeatedly purified by silica gel and HPLC column chromatography to afford EFL 3 (400 mg). EFL 3  Metalaxyl (98%) was purchased from Aladdin (Shanghai, China). EFL 3 and metalaxyl were dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution of 10 mg/mL. Other chemicals and reagents of analytical grade were purchased from Solarbio (Beijing, China).

P. capsici Isolates and Culture Conditions
Isolates (n = 105) of P. capsici from pepper, tomato, eggplant, and cucumber were collected from different areas of Jiangsu, Anhui, and Shandong Provinces in 2020 and 2021 (Table S1). The sampling fields had never been exposed to metalaxyl before, and they were at least 20 km apart from each other. In each region, samples (leaf, stem, fruit, or root) with typical symptoms of P. capsici infection were collected and cut into small pieces (3 × 3 mm). All pieces were surface sterilized using 2% NaClO (v/v), washed with sterilized distilled water, and then placed on V8 juice agar plates and incubated for 4 days at 25 • C. P. capsici isolates were identified by colony morphology, sporangium sharp, and sequence analysis [36]. Among the 105 P. capsici isolates, a metalaxyl-resistant strain (TA54, isolated from a host pepper plant in Tai'an, Shandong Province, China) was selected for studies on inhibitory activity and EFL 3 mechanism.

Determination of Baseline Sensitivity of P. capsici to EFL 3
The baseline sensitivity of P. capsici to EFL 3 was determined using the mycelial growth inhibition method [26]. The test P. capsici isolates (n = 105) were cultivated on V8 juice agar plates at 25 • C for 4 days to generate new mycelia. Next, fresh mycelial plugs were cut and incubated on V8 juice agar plates supplemented with 0, 1.25, 2.5, 5, 10, or 20 µg/mL of EFL 3 . After incubation for 4 days, the colony diameters were measured and growth inhibition rates were calculated. The median effective concentration (EC 50 ) values were calculated based on linear regression of the colony diameter on log-transformed EFL 3 concentration [37].
The baseline sensitivity of P. capsici to EFL 3 by spore germination was determined as previously reported [26]. The test P. capsici isolates (n = 105) were again cultivated on V8 juice agar plates at 25 • C for 4 days to generate new mycelia. Then, 10 fresh mycelial plugs were cut and incubated on a plate that contained 10 mL of V8 juice medium. After incubation for 2 days at 25 • C, the mycelia were washed and resuspended in sterilized tap water in the dark at 25 • C for 1 day for sporangial formation. Then, the cultures were transferred to 4 • C for 1 h followed by incubation at 25 • C to release zoospores from sporangia. Zoospore suspensions (1 × 10 5 spores/mL) were exposed to different concentrations of EFL 3 and then incubated at 25 • C in the dark for 12 h. Germinated spores were counted at three random sites on each plate under a microscope. The spore germination inhibition rate was calculated. EC 50 values were calculated based on linear regression on log-transformed EFL 3 concentration [38]. The bioassay data were obtained from three independent biological replicates.

OM and TEM Observations
The effects of EFL 3 on the mycelial morphology and ultrastructure of P. capsici were observed using OM and TEM observations [39]. Fresh mycelial plugs of LT263 (standard strain of P. capsici) and TA54 were cut and incubated on V8 juice agar plates supplemented with 0, 5, or 10 µg/mL of EFL 3 . After incubation for 2 days, the mycelia were taken from the periphery of the colonies and placed on glass slides. The P. capsici mycelial morphology was observed, and images were obtained using OM. For TEM, the samples were fixed in 4% (v/v) glutaraldehyde at 4 • C for 6 h and dissolved in 1% (v/v) osmium tetroxide at room temperature for 2 h. Samples were then subjected to a graded ethanol series (30,50,70,90, and 100% v/v) for 15 min at each stage for dehydration and embedded in Epon resin. After that, the samples were cut into ultrathin sections followed by staining with 2% (v/v) uranyl acetate and lead citrate. The mycelial ultrastructure of P. capsici was observed and photographed using a transmission electron microscope (Talos-F200C, Thermo Fisher Scientific, Waltham, MA, USA). Experiments were performed with three biological replicates, each with three technical replicates.

Effect of EFL 3 on Cell Membrane Permeability of P. capsici
The relative conductivity of P. capsici mycelia was measured following the conditions reported before [40]. First, 10 fresh mycelial plugs of LT263 and TA54 were cut and incubated into a flask that contained 100 mL of V8 juice medium. After incubation for 2 days at 25 • C with shaking at 175 rpm, EFL 3 at final concentrations of 0, 1.25, 2.5, 5, or 10 µg/mL was added to the flasks. After incubation for another day at 25 • C with shaking at 175 rpm, the mycelia were washed and resuspended in sterilized distilled water. Afterwards, the conductivity of the suspension was measured with a conductivity meter (DDS-11A, Shanghai Leici Instrument Inc., Shanghai, China) at 0, 10, 20, 40, 80, 100, and 120 min. After 120 min, all samples were boiled, and the final conductivity measurement was made. Relative conductivity (%) = (conductivity/final conductivity) × 100.
The effects of EFL 3 on MDA and glycerol contents in P. capsici mycelia were evaluated as described by Wang et al. [41]. Mycelia were cultured as described above. After being collected and washed with sterilized distilled water, mycelia samples were ground with liquid nitrogen and dissolved in PBS (pH 7.2). The supernatant was collected by centrifugation at 5000× g for 20 min. The MDA and glycerol contents of P. capsici mycelia were measured using MDA (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and glycerol (Beijing Applygen Technologies Inc., Beijing, China) detection kits. Three biological replicates were obtained.

Effect of EFL 3 on P. capsici Cell Wall and Cell Membrane Integrity
The effect of EFL 3 on cell wall integrity was measured as described before [26,42]. In brief, fresh mycelial plugs of LT263 and TA54 were cut and incubated into a plate containing 10 mL of V8 juice medium. After incubation for 1 day at 25 • C, EFL 3 at final concentrations of 0, 5, or 10 µg/mL was added to the plates. After incubation for another day at 25 • C, the mycelia were washed with PBS (pH 7.2) and stained with 10 µL of 1% (v/v) CFW and 10 µL of 10% (v/v) KOH for 1 min. The mycelia were then washed with PBS again and observed under a fluorescence microscope (LSM880, Carl Zeiss, Jena, Germany). The effect of EFL 3 on cell membrane integrity was detected using PI staining. Mycelia were cultured as described above. After incubation with different concentrations of EFL 3 for 1 day, the mycelia were washed with PBS and then stained with 50 µg/mL PI for 20 min in the dark. After that, the mycelia were rinsed with PBS again and observed under a fluorescence microscope. The experiment was repeated three times.

Effect of EFL 3 on P. capsici Phospholipid and Cellulose Contents
The effect of EFL 3 on phospholipid content was measured following the methods reported before [22]. Mycelia were cultured as described above in Section 2.6. After being collected and washed with sterilized distilled water, mycelia samples were ground with liquid nitrogen. Subsequently, 10 mg mycelia per sample were collected and resuspended in the assay buffer. The phospholipid content of P. capsici mycelia were measured using a phospholipid assay kit (Sigma-Aldrich, St. Louis, MO, USA). The effect of EFL 3 on cellulose content was also determined. Mycelia were cultured and collected as described above. After being ground with liquid nitrogen, 0.3 g of mycelia per sample were collected and resuspended in the extraction buffer. The cellulose content of P. capsici mycelia were measured by a cellulose assay kit (Solarbio, Beijing, China). The experiment was repeated three times.

Control Efficacy of EFL 3 against P. capsici
The control efficacy of EFL 3 against P. capsici was first tested on tomato leaves as reported before [41]. Tomato leaves (Solanum lycopersicum L. cv. 'Hongbaoshi') were detached from the tomatoes at the four-to six-leaf stage. Leaves with uniform size, color, and shape were selected after surface sterilization with 2% (v/v) NaClO. Following surface sterilization, tomato leaves were sprayed with EFL 3 at final concentrations of 0, 50, 100, and 200 µg/mL or metalaxyl at 200 µg/mL. For protective efficacy determination, the leaves were inoculated with fresh mycelial plugs after being sprayed with EFL 3 or metalaxyl dilutions for 24 h. For curative efficacy determination, the leaves were first inoculated with fresh mycelial plugs. Then, after 24 h, the surface of each tomato leaf was sprayed with the drugs. Leaves within the same treatment were placed in one sealed plastic container and incubated at 25 • C and a long-day photoperiod. After inoculation for 4 days, the lesion area on the leaves was measured and calculated. Each group consisted of 15 leaves, and the experiment was performed in triplicate. Control efficacy = (lesion area of water treatment − lesion area of drug treatment)/lesion area of water treatment.
Given that P. capsici is a soilborne pathogen, to further clarify the control efficacy of EFL 3 against P. capsici, pot experiments were carried out as described by Li et al. [43]. Pepper seedlings (Capsicum annuum L. cv. 'Sujiao 5 ) at the four-to six-leaf stage were obtained from vegetable fields in Nanjing, Jiangsu Province, China. Zoospore suspensions (1 × 10 5 spores/mL) of LT263 and TA54 were prepared as described above in Section 4.4. Pepper seedlings were sprayed with EFL 3 at final concentrations of 0, 100, and 200 µg/mL. A sterilized needle was used to prick the stem base of each pepper seedling to make a wound site. For protective efficacy determination, the wound site was inoculated with 5 µL of zoospore suspensions after being sprayed with EFL 3 dilutions for 24 h. For curative efficacy determination, the seedlings were first inoculated with zoospore suspensions. Then, after 24 h, the surface of each pepper seedling was sprayed with the treatments. Seedlings within the same treatment were placed in a growth chamber and incubated at 23-28 • C and a long-day photoperiod. After inoculation for 10 days, the disease severity was recorded using the crown rot system scale (0 to 5, Figure S5). The disease index = Σ(scale × plant numbers). Each group consisted of 20 seedlings, and the experiment was performed in triplicate. Control efficacy = (disease index of water treatment − disease index of drug treatment)/disease index of water treatment.

Statistical Analysis
Data are shown as the mean ± standard error (SE). All statistical analyses were carried out using SPSS software (v14.0 SPSS Inc., Chicago, IL, USA). The significance of the difference between treatments was determined using Fisher's least significant differences tests at p < 0.05 level.

Conclusions
In conclusion, we have demonstrated that EFL 3 exhibits strong inhibitory activity against mycelial growth and spore germination of P. capsici in vitro and that it is also effective for controlling diseases caused by P. capsici in vivo. The antifungal mechanism of EFL 3 may be attributed to its destructive potential in the cell membrane and cell wall via the inhibition of phospholipid and cellulose biosynthesis. However, further molecular studies are required to establish the molecular mechanisms by which EFL 3 negatively impacts P. capsici.